Project summary Reinforcing effects of alcohol are likely to be triggered by low doses of ethanol consumed once a subject is getting acquainted with alcoholic beverages. Moreover, it might be difficult to decipher mechanisms involving effects of high doses of alcohol without understanding mechanisms underlying effects of low doses. We have initiated mapping activity of immediate early genes in rodent brains almost 20 years ago. These initial studies found a number of brain regions in which a low dose of alcohol either induced c-Fos expression or suppressed basal or novelty- and stress-induced c-Fos expression. Subsequently, we also mapped brain regions in which c-Fos was induced after mice voluntarily consumed low doses of alcohol. However, these early studies were hampered by several problems with methodology at that time. These problems included: 1) The studies were performed during the bright phase of the circadian cycle, where activity and basal c-Fos expression is low, making it difficult to detect inhibition of neural activity, 2) Once staining was performed it was impossible to reuse the stained slices limiting double-labeling approaches and ability to identify neurochemical nature of subpopulations of neurons, 3) The peak time of c-Fos protein expression is relatively broad, therefore, c-Fos activation reflected not only effects of alcohol, but also presumably alcohol- independent behavior prior or after actions of alcohol. Recently developed technologies and approaches allow overcoming these technical difficulties. This exploratory grant proposes to use two comprehensive technologies to identify neuronal populations regulated by voluntary self-administration of low doses of alcohol in mice. This goal will be achieved in two Specific Aims. In Aim 1, we will create 3-dimentional maps of changes in activity of neurons following consumption of low doses of alcohol using the CLARITY technique. In Aim 2, we will create time-sensitive maps of changes in activity of neurons following consumption of low doses of alcohol using fluorescence-in situ-hybridization.